SARA is one of the most important metabolic disorders in ruminant production, which has a certain negative impact on rumen fermentation, animal welfare, productivity and farm profitability [30–32]. At present, time for pH below optimal level is used as a critical indicator to determine the severity of SARA. However, there are no uniform criteria for the rumen pH threshold and duration below a threshold. It has been reported that the continuous decline in pH threshold ranging from 5.8 to 5.5 exceeds 3 h/d is defined as SARA [33–35]. Deleterious effects of SARA, such as the increase of LPS concentrations in rumen and acute phase proteins in blood [14], are often observed with pH critical value depression below 5.6 for at least 3 h [5, 7, 36]. Kleen et al. [28] recommended pH 5.5 as the threshold for SARA. Based on the above references, we decided pH 5.5 as the threshold of SARA incidence. Among all goats, maximun pH, minimum pH, mean rumen pH, duration pH below 5.8 and 5.5 range from 6.91 to 6.24, 5.74 to 5.29, 6.14 to 5.37 and from 1.56 to 7.35 and 0 to 4.71 h/d, respectively, without a drop in ruminal pH below 5.2. when the dietary NFC/NDF was 2.66, indicating that SARA model was induced successfully of dairy goats in the current study.
Due to the structural differences between the epithelium of rumen and large intestine epithelium, and the hindgut lacks natural defense against SARA, it would be easier compromised by microbes and their products. Argenzio and Meuten [37] showed that acetic acid (0.1 M) had a time- and pH dependent ability to damage the colonic epithelium in pigs. In production practice, intestinal epithelial barrier dysfunction is frequently found in high-yield ruminants due to long-term feeding of high-grain diet. In this study, our data demonstrated that a profound structural disruptions of colonic epithelium was observed in SARA goats depicted by electron density of TJ decreased, intercellular space widened and mitochondria swelled. In contrast, histological structure and the intercellular ultrastructure were normal in the colonic epithelium of control goats. The results of the present study are in accordance with previous studies conducted in high grain-fed goat omasum [38], goat colon and cecum [39, 40], indicating the disruption and expansion of tight junction in the hindgut tissues. As previous reported on the rumen epithelial barrier [21, 41–43], grain-induced SARA can disrupt rumen epithelial structure and integrity in dairy cows and goats, resulting in breakdown of rumen epithelial barrier function.
The integrity of the intestinal epithelial barrier plays a central role in maintaining the digestive system and even the entire animal health, which is damaged or dysfunctional can increase epithelial permeability [44]. The ussing chamber was first proposed when studying the ion transfer of epithelial tissues. Its main function is to detect changes in the electric current signal of ion channel of the whole cell membrane through microelectrodes to reflect the intestinal epithelial permeability or drug absorption [45, 46]. This technology provides a short-term culture method for isolated tissues, which can truly reflect the real-time conditions of the animal's intestinal environment and is simple to operate. To date, it has become the main method to study the permeability of gastrointestinal tract by detecting the electrophysiological parameters to reflect the activity of epithelial tissue and the ratio of isotopic or fluorescein labeled flow-through epithelium[25, 47] .
The increase of Isc, as a measure for active epithelial ion transfer, as well as Gt, as a measure of passive ion “leakage” means an increase of both transport capacity and permeability cross the epithelium. PD reflects the activity of intestinal epithelium. Previous studies have suggested that high grain diets can increase the Isc and Gt of ruminal epithelium, and flow rate of marker molecules of different sizes such as HRP and FITC, leading to weakened barrier function [11]. Penner et al. [48] reported that a mild episode of SARA did not negatively affect epithelial barrier function in the short term. However, with the increase of acidification in vitro, a decrease of barrier function was found. In the present study, we monitored intestinal permeability in terms of Isc, Gt of the colonic epithelium and flux of FD4. The flux of intact FD4 across the intestinal epithelium occurs mainly through paracellular pathways [49, 50]. Our results indicated that a concurrent increase of the Isc, Gt and flux of intact FD4 across the colonic epithelium in SARA goats was observed, which showed that SARA induced higher epithelial permeability and barrier dysfunction. These results are in good agreement with the results of Sun et al [16] from this same experiment, who reported that SARA increased paracellular permeability of ruminal epithelium and compromised ruminal barrier. Therefore, our results combined with previous publications suggested that SARA not only impaired rumen epithelial barrier function of dairy goats, but also disrupted the integrity of the intestinal epithelium, resulting in an increase in permeability of colonic epithelium.
In the present study, We also used blood biochemical indicators to measure the barrier function of intestinal epithelium. Similar to the study of Andersen et al. [51] and Khafipour et al. [4], grain-induced SARA increased the concentration of LPS in peripheral blood. When the intestinal permeability increased abnormally due to some sort of disruption, allowing a large amount of LPS, D-lactic acid and DAO in the lumen to enter the blood circulation system. SARA could provide a stress on the hindgut epithelium via increasing the acidity and the LPS concentration of digesta. However, previous studies indicated that SARA did not increase LPS in peripheral blood [5, 7], which were inconsistent with the results of our study. Lactation can pose a stress on the ruminants and it may be one of the reasons for the difference [5]. In addition, our data also suggested that SARA significantly increased plasma D-lactic acid content and DAO activity. LPS, D-lactic acid and DAO in plasma have been considered as circulating markers for reflecting the degree of intestinal barrier injury [52, 53]. Thus, the changes of these indicators further suggested that SARA compromised the barrier function of the intestinal epithelium in dairy goats.
TJ is the most important connection between epithelial cells and also the key to form the intestinal mucosal barrier, which is mainly composed of TJ proteins [54]. Numerous studies have demonstrated the enrichment or defect of TJ proteins are causally related to a variety of human diseases, indicating that TJ proteins serve crucial role in human physiology [55–57]. In this study, with the disruption and expansion of intercellular TJs morphology, expression of Claudin-1 and Occludin were markedly up-regulated in the colonic epithelium of dairy goats during SARA, while no significant difference was observed in the expression of Claudin-4, Claudin-7 and ZO-1.
Upregulation of key TJ proteins would be expected to result in an increase of barrier function, but this was not the case in our study. The claudins comprise a multigene family, and the different claudins have diverse functions depending on cell type and the host organism [58]. Poritz et al [59] revealed that Claudin-1 doesn't localize to the TJ and may not contribute to the barrier function. The presence of Claudin-1 in the nucleus and cytoplasm may demonstrate that it has other functions. As previous reports, Claudin-1 expression has been confirmed to be upregulated in most types of malignancies, such as colorectal cancer [60–62]. Therefore, we speculate that the upregulation of Claudin-1 did not lead to an increase in intestinal epithelial barrier function. Occludin has a transmembrane region and may play a role in determining the function and structure of the paracellular barrier. In the present study, the increase of Occludin in colonic epithelium was basically consistent with the results of studies by Pederozoli et al.[63] and Tao et al. [41]. Reasons for the difference may be related to severity of SARA challenge, experimental animals, different dietary structures and the degree of damage to the intestinal epithelium. However, a recent study demonstrated that occludin depletion in intestinal epithelial cells in vitro and in vivo leads to an increase in paracellular flux of larger-sized molecules, suggesting that occludin plays a crucial role in the maintenance of intestinal epithelial TJ barrier function, through the large-channel TJ pathway [64], which is inconsistent with the results of the present study. Therefore, the true causal relationship between TJ structural disruption and Occludin increase in the colonic epithelium of dairy goats suffered from SARA is unclear and needs further study.